Biomimetic GBM-targeted drug delivery system boosting ferroptosis for immunotherapy of orthotopic drug-resistant GBM

Background Clinical studies have shown that the efficacy of programmed cell death receptor-1/programmed cell death ligand-1 (PD-1/PD-L1) inhibitors on glioblastoma (GBM) is much lower than what is expected because of the low immunogenicity of GBM. Ferroptosis of cancer cells can induce the maturation of dendritic cells (DC cells) and increase the activity of T cell. The activated T cells release IFN-γ, which subsequently induces the ferroptosis of cancer cells. Thus, the aim of this paper is to set up a new GBM-targeted drug delivery system (Fe3O4-siPD-L1@M-BV2) to boost ferroptosis for immunotherapy of drug-resistant GBM. Results Fe3O4-siPD-L1@M-BV2 significantly increased the accumulation of siPD-L1 and Fe2+ in orthotopic drug-resistant GBM tissue in mice. Fe3O4-siPD-L1@M-BV2 markedly decreased the protein expression of PD-L1 and increased the ratio between effector T cells and regulatory T cells in orthotopic drug-resistant GBM tissue. Moreover, Fe3O4-siPD-L1@M-BV2 induced ferroptosis of GBM cells and maturation of DC cell, and it also increased the ratio between M1-type microglia and M2-type microglia in orthotopic drug-resistant GBM tissue. Finally, the growth of orthotopic drug-resistant GBM in mice was significantly inhibited by Fe3O4-siPD-L1@M-BV2. Conclusion The mutual cascade amplification effect between ferroptosis and immune reactivation induced by Fe3O4-siPD-L1@M-BV2 significantly inhibited the growth of orthotopic drug-resistant GBM and prolonged the survival time of orthotopic drug-resistant GBM mice. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12951-022-01360-6.


Background
Glioblastoma (GBM) is an aggressive intracranial malignant tumor with high mortality and morbidity, accounting for 80% of malignant tumors in central nervous system (CNS). The overall median survival for GBM patients is only about 15 months [1,2]. At present, surgical resection followed by radiotherapy and temozolomide (TMZ) chemotherapy is considered to be the basic treatment for patients with newly diagnosed GBM [3][4][5]. However, it is frustrating that long-term use of TMZ in GBM patients inevitably leads to the overexpression of O 6 -methylguanine DNA methyltransferase (MGMT) in GBM cells, which results in the resistance of GBM cells to TMZ. Subsequently, the efficacy of TMZ is significantly reduced or even lost [6,7]. Therefore, it is an urgent need to find new treatment methods for TMZ-resistant GBM.
Immunotherapy, a very promising cancer treatment method, inhibits tumor growth and metastasis by inducing systemic and sustained immune response [8]. However, the efficacy of immunotherapy on GBM is much lower than what is expected. This is resulted from the following reasons. Firstly, as compared with other cancer such as non-small cell lung cancer, GBM in most cases shows a lower tumor mutational burden [9], resulting in lower immunogenicity of GBM cells and less recruitment of effector T cells (T eff cell) in GBM tissue [10]. Secondly, GBM cells usually recruit regulatory T cell (T reg cell) into GBM tissue by secreting chemokines such as colony stimulating factor 1 (CSF1), C-X-C Motif Chemokine Ligand 12 (CXCL12), C-X-C Motif Chemokine Ligand 1 (CXCL1) and granulocyte-macrophage colony stimulating factor (GM-CSF) [11]. T reg cell inhibits the function of T eff cell, subsequently reducing the generation of interleukin-2 (IL-2) and interferon-γ (IFN-γ) [12,13]. Finally, GBM cells are able to polarize anti-tumor M1 type microglia/macrophage into the immunosuppressive M2 type microglia/macrophage by secreting immunomodulatory cytokines [14,15]. M2 type microglia/macrophage also inhibits the function of T eff cell and promotes the progression of GBM by secreting cytokines such as interleukin-6 (IL-6), interleukin-10 (IL-10) and C-C Motif Chemokine Ligand 2 (CCL2) [16].
Ferroptosis is a form of iron-dependent cell death. The essences of ferroptosis are the over-load of Fe 2+ , depletion of glutathione (GSH) and the decrease of glutathione peroxidase (GPX4) [17,18]. Lipid oxides cannot be metabolized through GPX4. Subsequently, a large number of hydroxyl radicals are produced through Fenton reaction, leading to lipid peroxidation in cancer cells. This finally results in cancer cell death [19,20]. Many studies have shown that ferroptosis also leads to the maturation of DC cells in cancer tissue in vivo, and the matured DC cells present antigen to T lymphocytes to activated T eff [21,22]. Moreover, PD-1/PD-L1 inhibitor is able to activate T eff cell to secrete IFN-γ [23]. IFN-γ secreted by activated T eff cell inhibits the cysteine transporter (xCT) and subsequently prevents the cysteine from being taken up by cancer cells, resulting in the reduction of GSH synthesis in cancer cells. Then, ferroptosis is significantly enhanced in turn [24][25][26]. In theory, ferroptosis inducer and PD-1/PD-L1 inhibitor can mutually enhance each efficacy when they are simultaneously used to treat GBM. siRNA shows high specificity and low toxicity in cancer treatment [27]. It has potential application value to interfere PD-L1 protein synthesis in drug-resistant GBM cells by using siPD-L1. However, lack of suitable siPD-L1 delivery vector and easy degradation of siPD-L1 in blood circulation are the main obstacles that limit the application of siPD-L1 in the treatment of GBM [28,29]. As compared with other carriers, Fe 3 O 4 nanoparticle is a promising siPD-L1 carrier [30]. Firstly, Fe 3 O 4 nanoparticle displays good biocompatibility and biodegradability, and it is easily available. Fe 3 O 4 nanoparticle has been approved for clinic use by the Food and Drug Administration (FDA) [31]. Secondly, Fe 3 O 4 nanoparticle significantly increases the intracellular iron content especially Fe 2+ [32,33], which provides sufficient substrate for ferroptosis in drug-resistant GBM cells. Last, Fe 3 O 4 nanoparticle shows super paramagnetism, which allows it to be directed delivery by an external magnetic field [34]. However, Fe 3 O 4 nanoparticle is difficult to cross blood-brain barrier (BBB) [35,36]. Recent studies have shown that GBM tissue can recruit microglia by secreting chemokines such as C-X3-C motif chemokine ligand 1 (CX3CL1) and CSF-1 [37][38][39]. In theory, microglia membrane coated Fe 3 O 4 nanoparticle can be recruited to drug-resistant GBM.
In this study, disulfide bonds were used to connect thiolated siPD-L1 and thiolated Fe 3 O 4 nanoparticles to increase the stability of siPD-L1 in blood circulation. Fe 3 O 4 nanoparticles connected with siPD-L1 (Fe 3 O 4 -siPD-L1) are further coated with microglial membrane (M -BV2 ) to form a biomimetic brain-targeted nanoparticle Fe 3 O 4 -siPD-L1@M -BV2 . After Fe 3 O 4 -siPD-L1@M -BV2 was taken up by orthotopic drug-resistant GBM cells, the disulfide bond between Fe 3 O 4 nanoparticles and siPD-L1 was broken by intracellular GSH, releasing siPD-L1 and inhibiting the protein expression of PD-L1 in orthotopic drug-resistant GBM cells [40]. Subsequently, T eff cell was activated to enhance the killing effect on drug-resistant GBM cell [41]. At the same time, Fe 3 O 4 -siPD-L1@M -BV2 facilitated the ferroptosis of drug-resistant GBM cells, which further activated T eff cell by improving the maturation of DC cells. Moreover, activated T eff cell enhanced the ferroptosis of drugresistant GBM cells in turn by secreting IFN-γ. Finally, there forms a cascade amplification effect between ferroptosis and immune activation in orthotopic drugresistant GBM tissue (Scheme 1).

Extraction of BV2 cell membrane
BV2 cells in logarithmic growth phase were collected and mixed with 3 mL low-osmotic lysate and 30 μL protease inhibitor. Then, BV2 cells suspension was immerged into liquid nitrogen. After BV2 cells suspension was frozen and thawed for 3 times, the cell lysate was centrifuged for 10 min at 4 ℃ (14,000 × g). The supernatant was discarded, and 3 mL sterilized deionized water was added into precipitate. The mixture was performed ultrasound for 2 min, and supernatant was collected by centrifuging mixture for 20 min at 4 ℃ (14,000 × g). Then BV2 cell membrane (M -BV2 ) was obtained by lyophilizing the supernatant.

Preparation of Fe 3 O 4 -siPD-L1@M -BV2
Thiolated siPD-L1 (150 μL, 0.264 mg/mL), H 2 O 2 (30%, 55 μL) and thiolated Fe 3 O 4 nanoparticles (120 μL, 5 mg/ mL) were added into enzyme-free EP tube, and the mixture was stirred for 1 h at room temperature to connect siPD-L1 with Fe 3 O 4 nanoparticles by disulfide bond. The reaction mixture was centrifuged for 10 min at 4 ℃ (9000 × g), and the supernatant was discarded. The precipitate was washed with DEPC water for 6 times to completely remove free siPD-L1. After that, precipitate was re-suspended into PBS buffer to get Fe 3 O 4 -siPD-L1 suspension. The mass ratio between thiolated Fe 3 O 4 and thiolated siPD-L1 was optimized by agarose gel electrophoresis. Scramble siPD-L1 was used to prepare Fe 3 O 4 -siNC nanoparticle by using the same method in the preparation of Fe 3 O 4 -siPD-L1. Finally, 3 mL sterilized deionized water containing 10 mg M -BV2 was added into Fe 3 O 4 -siPD-L1 suspension. After being performed ultrasonic for 1 min, the mixture solution was incubated at 37 ℃ for 10 min.
The selective uptake of Fe 3 O 4 -FAM@M -BV2 by GL261/TR cell, HT-22 cell, BV2 cell and RAW264.7 cell GL261/TR cells, HT-22 cells, BV2 cells and RAW264.7 cells in logarithmic growth phase were separately inoculated into different 24-well plates containing cover glass at density of 2 × 10 5 cells/mL and incubated at 37 ℃ for 24 h. Four cover glass inoculated with different cells were transferred into one well of 6-well plate, and 2 mL of fresh serum-free dulbecco's modified eagle medium (DMEM) containing Fe 3 O 4 -FAM@M -BV2 (the equivalent Fe 3 O 4 concentration was 200 μg/mL) was added into each well. Fe 3 O 4 -FAM was used as control. The cells were cultured for 1, 2 and 4 h, respectively. (1) The cells were collected and re-suspend in PBS. The uptake of Fe 3 O 4 -FAM@M -BV2 by GL261/TR cell, HT-22 cell, BV2 cell and RAW264.7 cell was detected by flow cytometer (Beckman, A00-1-1102, USA). (2) The cell culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 10 min. The cells were washed with PBS for 3 times. Then cells were stained with 4' ,6-diamidino-2-phenylindole (DAPI) solution (0.5 μg/mL) for 10 min. After the cells were washed with PBS for 3 times, the uptake of Fe 3 O 4 -FAM@M -BV2 by GL261/TR cell, HT-22 cell, BV2 cell and RAW264.7 cell was observed by LSCM.
The uptake mechanism of Fe 3 O 4 -FAM@M -BV2 by GL261/TR cell (1) Fresh serum-free DMEM containing Fe 3 O 4 -FAM@M -BV2 (the equivalent Fe 3 O 4 concentration was 200 μg/mL) was co-incubated with CX3CR1 antibody (1 μg/mL) for 2 h at 37 ℃, and then the mixture was added into a 24-well plate pre-inoculated with GL261/TR cells on a cover glass. The cell was culture at 37 ℃ for 4 h. (2) Fresh serum-free DMEM containing CX3CL1 antibody (1 μg/mL) was added into a 24-well plate pre-inoculated with GL261/TR cells on a cover glass. After co-incubation at 37 ℃ for 2 h, fresh serumfree DMEM containing Fe 3 O 4 -FAM@M -BV2 (the equivalent Fe 3 O 4 concentration was 200 μg/mL) was added into cell culture medium, and cell was cultured at 37 ℃ for 4 h.
(3) Chlorpromazine solution (10 μg/mL), colchicine solution (800 μg/mL), methyl-β-cyclodextrin solution (5 μg/ mL), and 2-deoxy-D-glucose (900 μg/mL) were added into 24-well plates pre-inoculated with GL261/TR cells on a cover glass. The cell was incubated at 37 ℃ for 2 h, and then fresh serum-free DMEM containing  h. 20 μL MTT solution (5 mg/mL) was added into each well and incubated at 37 ℃ for 4 h. The culture medium in well was discarded, and 150 μL DMSO was added into each well. The absorbance of each well was measured at 490 nm by enzyme linked immune-analyzer (Bio-Rad Laboratories, Inc. California, USA), and the cell survival rate was calculated.
MTT method was also used to illuminate whether Fe 3 O 4 -siPD-L1@M -BV2 could induce ferroptosis. Briefly, GL261/TR cells in logarithmic growth phase were inoculated into 96-well plates at density of 3 × 10 4 cells/mL per well and incubated at 37 ℃ for 24 h. The culture medium was replaced with fresh serum-free DMEM medium containing Fe 3 O 4 -siPD-L1@M -BV2 (equivalent Fe 3 O 4 concentration was 10, 20, 50, 100, 200 μg/mL) at the present of IFN-γ(10 ng/mL), ferrostatin1 (Fer-1, 10 μM) and deferoxamine (DFO, 100 μM). The cells were incubated at 37 ℃ for 48 h. 20 μL MTT solution (5 mg/mL) was added into each well, and cells were incubated at 37℃ for 4 h. The culture medium in well was discarded, and 150 μL DMSO was added into each well. The absorbance of each well was measured at 490 nm by enzyme linked immuneanalyzer, and the cell survival rate was calculated. concentration was 200 μg/mL. The concentration of IFNγ, Fer-1 and DFO was 10 ng/mL, 10 μM and 100 μM, respectively. Cells were incubated at 37 ℃ for 48 h. The cells were collected and washed twice with 1 × assay buffer. Then, 1 mL staining solution was used to re-suspended cells, and cell suspension was incubated at 37 ℃ for 20 min. After centrifugation (2000 × g, 5 min), the supernatant was discarded, and the cells were re-suspended in PBS. The living and dead cells was observed under a fluorescence microscope (Nikon, Japan). concentration was 200 μg/mL. The concentration of IFN-γ, Fer-1 and DFO was 10 ng/mL, 10 μM and 100 μM, respectively. The cells were incubated at 37 ℃ for 48 h. Then, cells were collected and proteins were extracted. Western blot was used to detect protein expression of GPX4 and xCT in GL261/ TR cells. (1) For detection ROS level, 1 mL of DHE dye solution (diluted 1000 times with serum-free DMEM medium) was added into each well. The cells were incubated at 37 ℃ for 1 h. Then cells were fixed with 4% paraformaldehyde and stained with DAPI solution (0.5 μg/ mL). After the cells were washed with PBS for 3 times, the ROS in GL261/TR cells was observed under fluorescence microscope. (2) For detection lipid peroxidation level, 1 mL of C11 BODIPY dye solution (diluted 1000 times with serum-free DMEM medium) was added into each well. The cells were incubated at 37 ℃ for 0.5 h. Then cells were fixed with 4% paraformaldehyde and stained with DAPI solution (0.5 μg/mL). After the cells were washed with PBS for 3 times, the lipid peroxidation (LPO) in GL261/TR cells was observed under fluorescence microscope.

Effects of Fe 3 O 4 -siPD-L1@M -BV2 on DC cell maturation in vitro
Femur and tibia of C57 male mice were isolated and immersed in 75% ethanol for 5 min. Then, femur and tibia were immersed in serum-free roswell park memorial institute 1640 medium (RPMI1640). The ends of the femur and tibia were cut off with scissors, and the bone marrow cells were rinsed out from the femur and tibia by using serum-free RPMI1640 medium. The culture medium containing bone marrow cells was filtered (70 μm, BioFIL). The filtrate was centrifuged (1000×g, 3 min), and supernatant was discard. The cells were resuspended with red blood cell lysate (R1010, Solarbio). After the cell suspension was placed at room temperature for 1.5 min, RPM1640 complete medium was added. The supernatant was discarded by centrifugation (1000×g, 3 min), and the cells were re-suspended by RPM1640 complete medium containing GM-CSF (20 ng/mL). The cells were inoculated into 6-well plates at a density of 3×10 6 cells/mL in each well (1 mL). 3 days later, 1 mL of RPMI1640 complete medium containing GM-CSF (20 ng/mL) was added into each well and cultured for another 2 days.
2 mL of cell culture medium containing and Fe 3 O 4 -siPD-L1@M -BV2 +Fer-1 (the equivalent Fe 3 O 4 concentration was 200 μg/mL, the concentration of IFNγ, Fer-1 and DFO was 10 ng/mL, 10 μM and 100 μM, respectively) was added into transwell donor chamber planted GL261/TR cells, respectively. After incubation for 6 h, donor chamber was transferred to a 6-well plate inoculated with DC cells at the bottom and cultured for 24 h. DC cells were collected and re-suspended with PBS. APC-CD11c antibody (0.2 mg/mL), FITC-CD80 antibody (0.5 mg/mL) and PE-CD86 antibody (0.2 mg/mL) were added into cell culture medium, and cell was incubated at 4 ℃ for 30 min in dark room. The cells were collected and re-suspended with 200 μL PBS. The proportion of CD11c + , CD86 + and CD80 + DCs was detected by flow cytometer.

Efficiency of Fe 3 O 4 -siPD-L1@M -BV2 transport across the in vitro BBB
bEnd3 cells in the logarithmic growth phase were inoculated into the transwell donor chamber at a density of 5 × 10 5 cells/mL per well. Serum-free DMEM medium was added into the recipient chamber, and bEnd3 cells were cultured at 37 ℃. The complete medium was replaced every two days. The resistance between transwell donor chamber and recipient chamber was measured by using a resistance meter. When the resistance value exceeded 200 Ω/cm 2 , the in vitro BBB model was regarded to be successful established [45].
The transwell donor chamber was transferred into a 24-well plate inoculated with GL261/TR cells at the bottom. 400 μL of fresh serum-free DMEM medium containing Besides, GL261/TR cells at the bottom of recipient chamber were fixed with 4% paraformaldehyde and then were stained with DAPI solution (0.5 μg/mL). After the cells were washed with PBS for 3 times, the uptake of Fe 3 O 4 -FAM@M -BV2 by GL261/TR cells was observed by LSCM after it penetrated in vitro BBB.

Effects of CX3CL1 and CSF-1 on the transport of Fe 3 O 4 -siPD-L1@M -BV2 across the in vitro BBB
(1) After the successful establishment of the in vitro BBB model, the transwell donor chamber was transferred into a 24-well plates inoculated with GL261/TR cells at the bottom, and 800 μL fresh serum-free DMEM was added into recipient chamber. (2) After the successful establishment of the in vitro BBB model, the transwell donor chamber was transferred into a 24-well plates without GL261/TR cells at the bottom, and 800 μL fresh serum-free DMEM containing CX3CL1 (200 ng/mL) or CSF-1 (100 ng/mL) was added into recipient chamber. After that, 400 μL of fresh serum-free DMEM containing Fe 3 O 4 -FAM@M -BV2 was added into transwell donor chamber (the equivalent Fe 3 O 4 concentration was 200 μg/mL). The 24-well plates without GL261/TR cell and chemokine was used as the control. After incubation for 4 h, the fluorescence intensity of the culture medium in recipient chamber was measured by fluorescence spectrophotometer.
After the successful establishment of the in vitro BBB model, transwell donor chamber was transferred into a 24-well plate inoculated with GL261/TR cells. (1) Fe 3 O 4 -FAM@M -BV2 (the equivalent Fe 3 O 4 concentration was 200 μg/mL) was incubated with fresh serumfree DMEM containing CX3CR1 or CSF-1R antibody for 2 h at 37 ℃, and then they were added into transwell donor chamber. 800 μL of serum-free DMEM was added into recipient chamber. (2) Fresh serum-free DMEM containing CX3CL1 or CSF-1 antibody was added into recipient chamber and incubated with GL261/TR cells at 37 ℃ for 2 h. After that, 400 μL of fresh serum-free DMEM containing Fe 3 O 4 -FAM@M -BV2 was added into donor chamber (the equivalent Fe 3 O 4 concentration was 200 μg/mL). The concentration of antibody was 1 μg/mL. Fe 3 O 4 -FAM@M -BV2 without incubation with antibody was used as control. After incubation for 4 h, the fluorescence intensity of the culture medium in recipient chamber was measured by fluorescence spectrophotometer.

Establishment of orthotopic drug-resistant GBM model in mice
Luciferase expressed GL261/TR cell (Luc-GL261/TR) in logarithmic growth phase were prepared as 2 × 10 7 /mL cell suspension. C57 mice were anesthetized and fixed on the operation table, and a micro-injector was inserted into the skull at the right front 2 mm of the intersection of sagittal suture and coronal suture. 5 μL of cell suspension was slowly injected into the brain with the brain stereotactic locator to establish orthotopic drug-resistant GBM model in vivo.

Distribution and pharmacokinetics of Fe 3 O 4 -siPD-L1@M -BV2 in orthotopic drug-resistant GBM mice
On the 10th day after plantation of Luc-GL261/TR cell, luciferase substrate was intraperitoneally injected (150 mg/kg). 5 min post injection, the mice were anesthetized with isoflurane. The GBM growth was observed by bioluminescence imaging (IVIS Lumina II, Caliper, USA), and the mice that the volume of orthotopic drug-resistant GBM did not meet the requirements were excluded.
The GBM-bearing mice were randomly divided into 4 groups. siPD-L1, Fe 3 O 4 -siPD-L1, Fe 3 O 4 -siPD-L1@M -BV2 and Fe 3 O 4 -siPD-L1@M -BV2 + magnet (Additional file 1: Fig. S1) were injected into GBM-bearing mice via tail vein (siPD-L1 was labeled by Cy5, equivalent siPD-L1 dose was 0.3 mg/kg). 6 h and 12 h later, in vivo bioluminescence imaging was used to observe the fluorescence distribution in the whole body of orthotopic drugresistant GBM mice. The integrated brain targeting efficiency was calculated with the following equation. The integrated brain targeting efficiency = (fluorescence intensity in brain organ/fluorescence intensity in whole body) × 100%. Blood, brain, heart, liver, spleen, lung and kidney tissues were collected at 0.08, 1, 3, 6, 12 and 24 h after drug administration. In vivo bioluminescence imaging was used to observe the distribution of Cy5 labeled Fe 3 O 4 -siPD-L1@M -BV2 in brain, heart, liver, spleen, lung and kidney. The brain tissue was immobilized in 4% paraformaldehyde for 24 h. After tissue was sectioned, DAPI was used to label the nuclei, CD31 antibody was used to label the tumor vessels, and the distribution of Fe 3 O 4 -siPD-L1@M -BV2 in drug-resistant glioma tissue was observed by LSCM. Fluorescence spectrophotometer was used to detect the Cy5 labeled Fe 3 O 4 -siPD-L1@M -BV2 concentration in plasma samples. The GBM tissue was isolation from brain tissue. The GBM tissue and normal brain tissue were respectively ground with 0.8 mL PBS buffer in an ice bath, and the tissue homogenate was centrifuged (9000 × g, 15 min, 4 ℃). The precipitation and supernatant were separated. (1) The concentration of Cy5 labeled Fe 3 O 4 -siPD-L1@M -BV2 in extracellular fluid of brain tissue was detected by fluorescence spectrophotometer. (2) The precipitation was ground with 0.6 mL RIPA cell lysate. The cell lysate was centrifuged (9000 × g, 20 min, 4 ℃). The intracellular Cy5 labeled Fe 3 O 4 -siPD-L1@M -BV2 concentration in GBM tissue was detected by fluorescence spectrophotometer.
(3) The intracellular content of Fe 2+ in GBM tissue was detected by using Perls stain kit. The precipitation was ground with 0.6 mL RIPA cell lysate. The cell lysate was centrifuged (9000 × g, 20 min, 4 ℃). 0.5 mL of supernatant was added into 0.5 mL NH 4 Fe(SO 4 ) 2 solution. The above mixture solution was shaken at room temperature for 30 min. Then, the absorbance value of mixture solution at 700 nm was detected by using UV spectrophotometer. After that, dilute nitric acid was added into mixture solution, and the absorbance value of mixture solution at 700 nm was detected again by using UV spectrophotometer. The content of Fe 2+ can be calculated according to the change of absorbance value.

The therapeutic effect of Fe 3 O 4 -siPD-L1@M -BV2 on orthotopic drug-resistant GBM in mice
On the 10th day after plantation of Luc-GL261/TR cell, luciferase substrate was intraperitoneally injected (150 mg/kg). 15 min later, the mice were anesthetized with isoflurane. The GBM growth was observed by bioluminescence imaging, and the mice that the volume of orthotopic drug-resistant GBM did not meet the requirement was excluded.

Statistical analysis
All data are expressed as mean ± standard deviation. The statistics analysis of each group was performed by using one-way ANOVA with SPSS 26.0 statistical software. p < 0.05 was considered statistically significant.

Transport of Fe 3 O 4 -FAM@M -BV2 across in vitro BBB
Within 12 h when drug is administered, there was no significant difference in resistance values between donor chamber and recipient chamber. It indicated   siPD-L1 in orthotopic drug-resistant GBM tissue increased successively in comparison with naked siPD-L1 and Fe 3 O 4 -siPD-L1. Moreover, the content of siPD-L1 in orthotopic drug-resistant GBM tissue was significantly higher than that in normal brain tissue (Fig. 6E-G). As compared with the Fe 3 O 4 -siPD-L1 group, the intracellular level of siPD-L1 in drug-resistant GBM tissue was significantly increased in Fe 3 O 4 -siPD-L1@M -BV2 group and Fe 3 O 4 -siPD-L1@M -BV2 + magnet group (Fig. 6H-J). The intracellular level of Fe 2+ in orthotopic drug-resistant GBM tissue was significantly increased in Fe 3 O 4 -siPD-L1@M -BV2 group and Fe 3 O 4 -siPD-L1@M -BV2 + magnet group as compared with that in Fe 3 O 4 -siPD-L1 group (Fig. 6K).

The inhibitory effect of Fe 3 O 4 -siPD-L1@M -BV2 on the growth of orthotopic drug-resistant GBM in mice
In vivo bioluminescence imaging was used to dynamic observe the growth of orthotopic drug-resistant GBM   H&E staining of orthotopic drug-resistant GBM tissue presented a long oval shape and a large number of pathological nuclear mitosis in normal saline, TMZ and Fe 3 O 4 treated group. Fe 3 O 4 -siPD-L1@M -BV2 decreased the number of heteromorphic nuclear cells and nuclear division in orthotopic drug-resistant GBM tissue. The nucleus was regular spherical and the intercellular space became blurred or even disappeared in Fe 3 O 4 -siPD-L1@M -BV2 + magnet treated group (Fig. 7E) Secondly, Fe 3 O 4 -siPD-L1@M -BV2 decreased the protein expression of PD-L1, x-CT and GPX4 in orthotopic drugresistant GBM tissue in dose-dependent manner. As compared with Fe 3 O 4 -siPD-L1, Fe 3 O 4 -siPD-L1@M -BV2 reduced the protein expression of PD-L1, xCT and GPX4 more strongly. The protein expressions of PD-L1, xCT and GPX4 in orthotopic drug-resistant GBM tissue were less in Fe 3 O 4 -siPD-L1@M -BV2 + magnet group than that in Fe 3 O 4 -siPD-L1@M -BV2 group (Fig. 8A, B). At the same time, immunofluorescence staining was also used to investigate the effects of Fe 3 O 4 -siPD-L1@M -BV2 on the protein expression of GPX4 and PD-L1 in orthotopic drug-resistant GBM tissue, and the results were consistent with western blot experiment (Fig. 8C, Additional file 1: Fig. S14). In addition, Fe 3 O 4 -siPD-L1@M -BV2 dose-dependently reduced the content of GSH in orthotopic drug-resistant GBM tissue. The GSH content in orthotopic drug-resistant GBM tissue in Fe 3 O 4 -siPD-L1@M -BV2 + magnet group was lower in comparison with that in Fe 3 O 4 -siPD-L1@M -BV2 group (Fig. 8D). Meanwhile, Fe 3 O 4 -siPD-L1@M -BV2 dosedependently increased the ROS level in orthotopic drugresistant GBM tissue (Fig. 8C, E). Moreover, as compared with normal saline, Fe 3 O 4 -siNC and Fe 3 O 4 -siNC@M -BV2 also significantly inhibited the GPX4 expression, GSH and ROS level in orthotopic drug-resistant GBM tissue, indicating that Fe 3 O 4 -siNC and Fe 3 O 4 -siNC@M -BV2 induced ferroptosis and subsequently inhibited the growth of orthotopic drug-resistant glioma.

Preliminary safety evaluation of Fe 3 O 4 -siPD-L1@M -BV2 in orthotopic drug-resistant GBM mice
On the 24th day after plantation of Luc-GL261/TR cell, H&E staining showed that no obvious abnormal morphological changes were observed in the heart, liver, spleen, lung and kidney in all treatment groups (Fig. 10A). Biochemical analysis further showed that ALT and AST activities, BUN and CREA contents in mice serum in each treatment group were all within the normal range ( Fig. 10B-E).

Discussion
The PD-1/PD-L1 signaling pathway plays an important role in the tumor immune microenvironment [46]. The activation of PD-1/PD-L1 signaling pathway induces the apoptosis of the T eff cell. Blocking the PD-1/PD-L1 signaling pathway activates T eff cell, thus inhibiting tumor growth [47]. However, clinical data show that only less than 10% of patients with GBM response to immunotherapy because of the low immunogenicity of GBM [10,48]. It has been shown that ferroptosis of cancer cells release high-mobility group box 1 (HMGB1) in an autophagy dependent manner [49], which increases the immunogenicity of cancer cells and promotes the maturation of DC cells [50][51][52]. The matured DC cells presents antigen to T lymphocytes and activates innate and adaptive immunity.
The key features of ferroptosis are the overload of Fe 2+ , depletion of GSH, the decrease of GPX4 activity and the consequent lipid peroxidation of cell membrane [53]. The results of MTT assay showed that Fe 3 O 4 -siPD-L1@M -BV2 decreased GL261/TR cell activity in a concentrationdependent manner. However, the inhibitory effect of Fe 3 O 4 -siPD-L1@M -BV2 on GL261/TR cell activity was significantly reduced in the present of ferroptosis inhibitors. After GL261/TR cells were incubated with Clinical studies have shown that there is a large number of microglia in GBM tissue [54,55]. GBM tissue promotes the polarization of microglia toward anti-inflammatory M2 type [11]. Studies have also shown that PD-L1 antibody activate mice macrophages to secrete more TNF-α and IL-12, which induce polarization of macrophages toward a pro-inflammatory M1 type [56,57]. The in vivo experimental results showed that Fe 3 O 4 -siPD-L1@M -BV2 significantly increased the content of IFN-γ, TNF-α and IL-12 in orthotopic drug-resistant GBM tissue. The ratio between M1 type microglia and M2 type microglia in orthotopic drug-resistant GBM tissue was significantly increased by Fe 3 O 4 -siPD-L1@M -BV2 . The above results demonstrated that Fe 3 O 4 -siPD-L1@M -BV2 polarized M2 type microglia to M1 type microglia through increasing IFN-γ, TNF-α and IL-12 content in orthotopic drugresistant GBM tissue.
Improving the stability in blood circulation and targeting to GBM is the key point for siPDL-L1 to play its role in vivo. Pharmacokinetic results showed that Fe 3 O 4 -siPD-L1@M -BV2 markedly increased the stability of siPD-L1 in blood. In vivo bioluminescence imaging results indicated that the coating of microglia membrane improved the brain targeting of Fe 3 O 4 nanoparticles. LSCM observation indicated that Fe 3 O 4 -siPD-L1@M -BV2 selectively distributed in the orthotopic drug-resistant GBM tissue. In addition, as compared with Fe 3 O 4 -siPD-L1, the intracellular siPD-L1 in orthotopic drug-resistant GBM tissue was significantly increased after Fe 3 O 4 -siPD-L1@M -BV2 was administrated. Meanwhile, the content of Fe 2+ in orthotopic drug-resistant GBM tissue was also significantly increased by Fe 3 O 4 -siPD-L1@M -BV2 . Those results demonstrated that Fe 3 O 4 -siPD-L1@M -BV2 exhibited good targeting for orthotopic drug-resistant GBM tissue. siPD-L1 and Fe 2+ were simultaneously delivered to orthotopic drugresistant GBM by Fe 3 O 4 -siPD-L1@M -BV2 . Due to the paramagnetism of Fe 3 O 4 nanoparticle, the brain targeting rate and the accumulation of Fe 3 O 4 -siPD-L1@M -BV2 in orthotopic drug-resistant GBM tissue was further increased under external magnetic field.
The in vivo imaging showed that Fe 3 O 4 -siPD-L1@M -BV2 was mainly distributed in brain and liver, while less Fe 3 O 4 -siPD-L1@M -BV2 was distributed in kidney and other tissue after intravenous administration. The amount of Fe 3 O 4 -siPD-L1@M -BV2 in brain tissue reached its maximum value at 12 h after administration. The distribution of Fe 3 O 4 -siPD-L1@M -BV2 in liver and brain significantly decreased at 24 h after administration. The above results indicated that Fe 3 O 4 -siPD-L1@M -BV2 and Fe 3 O 4 -siPD-L1@M -BV2 + megnet did not accumulate in the liver and kidney after a single tail vein injection. On the 24th day after plantation of Luc-GL261/TR cell, H&E staining and biochemical analysis indicated when Fe 3 O 4 -siPD-L1@M -BV2 and Fe 3 O 4 -siPD-L1@M -BV2 + megnet were intravenously injected into orthotopic drugresistant GBM mice for 4 times within 12 days, they did not cause significant damage to heart, liver, spleen, lung and kidney in orthotopic drug-resistant GBM mice. However, this just was a preliminary safety evaluation. It is not certain yet if hepatorenal toxicity can be caused by Fe 3 O 4 -siPD-L1@M -BV2 and Fe 3 O 4 -siPD-L1@M -BV2 + megnet when its dose is greater than 1 mg/kg or more than 4 times of administration. This needs further study.
Western blot results showed that CX3CL1 and CSF-1 were highly expressed in GL261/TR cells, while CX3CR1 and CSF-1R were highly expressed in the membrane of microglia and surface of Fe 3 O 4 -siPD-L1 nanoparticles. In vitro studies showed that in the existence of chemokine CSF-1 and CX3CL1, the efficiency of Fe 3 O 4 -siPD-L1@M -BV2 penetration in vitro BBB was significantly improved, and the accumulation of Fe 3 O 4 -siPD-L1@M -BV2 in GL261/TR cells was also markedly increased. In contrast, CSF-1 antibody, CX3CL1 antibody, CSF-1R antibody and CX3CR1 antibody significantly reduced the efficiency of Fe 3 O 4 -siPD-L1@M -BV2 penetration in vitro BBB and its accumulation in GL261/TR cells. The results demonstrated that the interaction between chemokines (CSF-1 and CX3CL1) secreted by GL261/TR cells and receptors (CSF-1R and CX3CR1) on the microglia membrane promoted Fe 3 O 4 -siPD-L1@M -BV2 to penetrate BBB and accumulate in GL261/TR cells.

Conclusion
Fe 3 O 4 -siPD-L1@M -BV2 was actively delivered to orthotopic drug-resistant GBM cell through the interaction between microglia membrane and GBM cell.
Authors' contributions BL, DL, and SZ conceived the manuscript. BL wrote the first draft. DL and SZ revised the first draft. BL and QJ performed the experiments. YC, ML, BZ, and QM provided grouping suggestions. All of the authors have read and approved the final manuscript.

Funding
This work was financially supported by the National Natural Science Foundation of China (81872803, 82073775), Shaanxi Province Key Research and Development Projects of China (2021ZDLSF03-08) and Shaanxi Natural Science Foundation (2020JQ-458).